Bordetella outer-membrane protein antigens and methods of making and using the same

ABSTRACT

An isolated protein or peptide selected from the group consisting of  Bordetella  colonization factor A (BcfA) protein and antigenic fragments thereof is described, along with an isolated nucleic acid encoding the same, antibodies that bind to the same, methods of producing an immune response in a mammalian subject in need thereof by administering the proteins, peptides or antibodies, and pharmaceutical compositions comprising the same.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/498,537, filed Sep. 26, 2014, which is acontinuation of and claims priority to U.S. patent application Ser. No.12/680,823, filed Jul. 16, 2010, now issued as U.S. Pat. No. 8,877,201,which is a 35 U.S.C. §371 national phase application of PCT ApplicationPCT/US2008/012051, filed Oct. 23, 2008, and published in English on Jul.30, 2009, as International Publication No. WO 2009/094006, and whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/982,513, filed Oct. 25, 2007, the disclosure of each of which isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government Support under Grant No.NCR-2005-05000 from the USDA. The United States Government has certainrights to this invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 9151-102_ST25.txt, 38172 bytes in size, generated onJul. 15, 2010, and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention concerns antigens, formulations thereof, andmethods of using the same.

BACKGROUND OF THE INVENTION

Bordetellae are Gram-negative bacteria that colonize the respiratorytracts of humans and animals. Bordetella bronchiseptica and Bordetellapertussis are well-adapted pathogens of the human and animal respiratorytract, respectively. Bordetella pertussis infects only humans and causesthe acute respiratory disease known as whooping cough. It is estimatedthat 20-30% of adolescents and adults who have chronic cough lasting formore than one week are infected with B. pertussis. The current acellularvaccines, although effective against severe symptoms, are notparticularly effective in preventing the carrier state. Adult andadolescent carriers harboring B. pertussis in the nasopharynx areresponsible for the familial transmission of the bacteria to infants andyoung children, in whom the disease is severe and sometimes lethal.Thus, the continued presence of B. pertussis and B. parapertussis andthe resurgence of pertussis despite widespread vaccinations, make thedevelopment of efficacious vaccines a top priority.

B. bronchiseptica has a broad host range infecting a variety of animals.It typically establishes asymptomatic infections but can cause atrophicrhinitis in pigs, kennel cough in dogs, snuffles in rabbits andbronchopneumonia in guinea pigs. Animals continue to be carriers of B.bronchiseptica despite vaccinations and frequently shed bacteriaresulting in outbreaks among herds. Since B. bronchiseptica can causerespiratory disease in immunocompromized patients, vaccination of petsand food-producing animals with attenuated B. bronchiseptica may posehealth risks to these patients through zoonotic transmission. Thus,there is a need to develop acellular vaccines for immunizing animals.

SUMMARY OF THE INVENTION

This invention is based upon our identification of a gene, designated byus as BcfA (Bordetella colonization factor A) by a bioinformaticsapproach. We produced and purified BcfA-T7-tagged fusion protein from E.coli, and have raised anti-sera against the purified protein in rats.Western-blotting with anti-BcfA antibody indicated that BcfA islocalized to the outer membrane and that it is expressed duringBordetella infection of rats. By intranasal infection of rats, we haveshown that BcfA plays an important role in respiratory colonization ofB. bronchiseptica. We have also found that BcfA is expressed in recentclinical isolates of B. pertussis from human patients. Pilot experimentsconducted in the laboratory also provide evidence that anti-serumagainst BcfA is able to protect mice against subsequent challenge withB. bronchiseptica. These data indicate that BcfA is useful as a vaccineand that anti-BcfA serum has a protective effect in animals.

A first aspect of the invention is an isolated protein or peptideselected from the group consisting of Bordetella colonization factor A(BcfA) protein and antigenic fragments thereof. In some embodiments, theBcfA protein has the sequence of SEQ ID NO:2. In some embodiments, theprotein or peptide is an antigenic fragment of BcfA from 20 to 500 aminoacids in length. In some embodiments, the protein or peptide is anantigenic fragment of BcfA having the sequence given herein as SEQ IDNO:3 or an antigenic fragment comprising 10 or more contiguous aminoacids thereof.

A further aspect of the invention is an isolated nucleic acid thatencodes a protein or peptide as described herein. The nucleic acid mayin some embodiments be operatively associated with a promoter, and insome embodiments may be in a host cell that contains the nucleic acidand expresses the encoded protein or peptide.

A further aspect of the invention is a method of producing an immuneresponse in a mammalian subject in need thereof, comprisingadministering the subject a protein or peptide as described herein in anamount effective to produce an immune response in that subject (e.g., aprotective immune response to Bordetella infection, such as a Bordetellabronchiseptica or Bordetella pertussis infection).

A further aspect of the present invention is a composition comprising aprotein or peptide as described herein in a pharmaceutically acceptablecarrier.

A further aspect of the invention is an isolated antibody (e.g., amonoclonal antibody or polyclonal antibody) that binds to BcfA protein(e.g., a protein of SEQ ID NO:2). In some embodiments the antibody maybe coupled to a solid support or a detectable group.

A further aspect of the present invention is a composition comprising anantibody as described herein in a pharmaceutically acceptable carrier.

A further aspect of the present invention is a method of treating amammalian subject for a Bordetella infection (e.g., a Bordetellabronchiseptica or Bordetella pertussis infection), comprisingadministering the subject an antibody as described herein in a treatmenteffective amount.

A further aspect of the invention is a method of detecting Bordetella(e.g., Bordetella bronchiseptica or Bordetella pertussis) in abiological sample, comprising: contacting the sample to an antibody asdescribed herein; and then detecting the presence or absence of specificbinding of the antibody to the sample, the presence of specific bindingto the sample indicating the presence of Bordetella in the sample.

A still further aspect of the invention is the use of a protein,peptide, or antibody as described herein for the preparation of amedicament for carrying out a method of treatment as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence encoding BcfA protein.

FIG. 2A shows the amino acid sequence of full-length BcfA. FIG. 2B showsthe predicted 508 amino acid residue extracellular domain of BcfA. FIGS.2C-2L show fragments of the extracellular domain of BcfA.

FIG. 3 shows that Immunization with BcfA protects mice against B.bronchiseptica challenge.

DETAILED DESCRIPTION OF THE INVENTION

Subjects to be treated by the methods of the present invention aregenerally mammalian subjects, including but not limited to human,monkey, chimpanzee, ape, dog, cat, pig, rabbit, goat, cow, cattle,horse, etc. Subjects may be male or female and may be any age includingneonate, infant, juvenile, adolescent, adult, and geriatric subjects.

“Antibodies” as used herein refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonalor polyclonal and may be of any species of origin, including (forexample) mouse, rat, rabbit, horse, or human, or may be chimericantibodies. See, e.g., M. Walker et al., Molec. Immunol. 26, 403-11(1989). The antibodies may be recombinant monoclonal antibodies, forexample produced according to the methods disclosed in Reading U.S. Pat.No. 4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. Theantibodies may be humanized or chimeric antibodies. The antibodies mayalso be chemically constructed according to methods such as disclosed inSegal et al., U.S. Pat. No. 4,676,980.

“Antigenic fragment” of a protein (e.g., BcfA) as used herein is anyportion of the protein that, when administered in accordance with themethods described herein, elicits, in a subject, an immune response thatis either a fragment-specific or specific for the protein from which thefragment was obtained. The immune response can be either a humoral or acell-mediated response. Antigenic fragments are known. See, e.g., U.S.Pat. No. 7,101,987; see also U.S. Pat. Nos. 7,270,816; 7,211,411;7,163,685; and 7,151,082. Antigenic fragments can be of any suitablelength (e.g., from 10, 12 or 20 contiguous amino acids up to 50, 100 or200 contiguous amino acids or more) and generated by known techniquessuch as epitope mapping. (e.g., a fragment that includes an epitoperegion as described below).

1. Antigens

The present invention includes B. bronchiseptica compositions composedof one or more B. bronchiseptica antigens against which it is desired togenerate an immune response. The use of bacterial antigens in theproduction of antigen compositions and vaccines is well-known in the artand described in, for example, U.S. Pat. No. 7,255,867.

Compositions of the invention may be composed of BcfA (SEQ ID NO:2), theextracellular domain of BcfA (SEQ ID NO:3), or fragments or epitopesthereof. The instant vaccine can be a monovalent vaccine or multi-valentvaccine. Multi-valent vaccines generally include more than one type ofantigen and can be produced by mixing a number of different antigens.

The instant antigen(s) can be made using any conventional synthetic orrecombinant means. The amino acid sequence of an antigen for use in theinvention can be modified to include non-naturally occurring amino acidsor to increase the stability of the compound. When the antigen isproduced by synthetic means, such amino acids may be introduced duringproduction. The antigen may also be modified following either syntheticor recombinant production.

The antigen for use in the invention may also be produced using D-aminoacids. In such cases, the amino acids will be linked in reverse sequencein the C to N orientation. This is conventional in the art for producingsuch peptides. A number of side chain modifications are also known inthe art and may be made to the side chains of the antigen for use in thepresent invention. Such modifications include, for example,modifications of amino acids by reductive alkylation by reaction with analdehyde followed by reduction with NABH₄, amidination withmethylacetimidate or acylation with acetic anhydride.

An antigen for use in the invention can be produced in large scalefollowing purification by high pressure liquid chromatography (HPLC) orother techniques after recombinant expression as described herein.

Polynucleotides to produce an antigen for use in the invention caninclude DNA or RNA. They may also be polynucleotides which includewithin them synthetic or modified nucleotides. A number of differenttypes of modifications to polynucleotides are known in the art. Theseinclude methylphosphate and phosphorothioate backbones, addition ofacridine or polylysine chains at the 3′ and/or 5′ ends of the molecule.Although the techniques mentioned herein are generally well-known in theart, reference may be made in particular to Sambrook and Russell (2001)Molecular Cloning: A Laboratory Manual, 3^(rd) Edition, CSHL Press.

An antigen for use in the present invention can be produced byrecombinant means by providing a polynucleotide encoding the antigenand, where appropriate, encoding any desired flanking sequences underthe control of a promoter and other required sequences. Such apolynucleotide is generally provided in the form of an expressionvector.

Such vectors can be transformed into a suitable host cell to provide forexpression of an antigen of the invention. Thus, an antigen for useaccording to the invention can be obtained by cultivating a host celltransformed or transfected with an expression vector as described aboveunder conditions to provide for expression of the antigen, andrecovering the expressed antigen.

The vectors may be, for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the said polynucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid. Promoters and other expression regulation signals may beselected to be compatible with the host cell for which the expressionvector is designed.

Host cells transformed (or transfected) with the polynucleotides orvectors for the replication and expression of polynucleotides of theinvention will be chosen to be compatible with the said vector andpreferably will be bacterial, e.g., E. coli. Alternatively they may becells of a human or animal cell line such as CHO or COS cells, or yeastor insect cells. The cells may also be cells of a non-human animal suchas a sheep or rabbit or plant cells.

2. Antigen Compositions

An antigen composition of the present invention can also include one ormore adjuvants. Adjuvants for use in the production of antigeniccompositions such as vaccines are well-known and routinely employed bythe skilled artisan. See, e.g., U.S. Pat. No. 7,183,402. For example,adjuvants for parenteral administration include aluminum compounds, suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate. The antigen is precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants, suchas RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteraladministration.

Adjuvants for mucosal administration include bacterial toxins, e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof such as a purified preparation ofnative cholera toxin subunit B (CTB). Fragments, homologs, derivatives,and fusions to any of these toxins are also suitable, provided that theyretain adjuvant activity. Preferably, a mutant having reduced toxicityis used. Suitable mutants are described, e.g., in WO 95/17211, WO96/06627, and WO 95/34323. Other adjuvants, such as a bacterialmonophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota,Salmonella typhimurium, or Shigella flexneri; saponins, or polylactideglycolide (PLGA) microspheres, are also be used in mucosaladministration.

Adjuvants useful for both mucosal and parenteral administrations includepolyphosphazene (WO 95/02415), DC-chol (3 b-(N—(N′,N′-dimethylaminomethane)-carbamoyl) cholesterol (U.S. Pat. No. 5,283,185 and WO96/14831) and QS-21 (WO 88/09336).

The compositions of the present invention may be administered by anysuitable route. The compositions can be formulated for delivery by amucosal, parenteral or transdermal route. Mucosal delivery routesinclude nasal, oral and oropharangeal routes, whereas parenteral routesinclude intramuscular, intraperitoneal, or subcutaneous injection.

Suitable binders and carriers may also be introduced into the presentcomposition depending on the type of formulation that is provided. Oralformulations typically may include excipients such as, for example,pharmaceutical grades of mannitol, lactose, starch, sodium saccharine,cellulose, and magnesium carbonate. In some embodiments, vaccination iscarried out by intranasal delivery of a liquid or spray.

The compositions are administrated in a manner compatible with thedosage formulation in such an amount as will be prophylacticallyeffective. The quantity to be administered depends on a number offactors. These include the subject to be treated, capacity of thesubject's immune system to synthesize antibodies and the degree ofprotection desired. Precise amounts of active ingredient required to beadministered may depend on the judgment of the practitioner. In general,the dose per subject may be 5 μg, 50 μg, or 250 μg, up to 10 mg or 100mg, per dose.

The compositions may be given in a single dose schedule or preferably ina multiple-dose schedule. A multiple-dose schedule is one in which aprimary course of vaccination may be with 1 or 2 up to 5 or 10 separatedoses, followed by other doses given at subsequent time intervalsrequired to maintain and/or reinforce the immune response, for example,at 1 to 4 months for a second dose and if needed, a subsequent dose(s)after several months.

3. Antibodies

Polyclonal antibodies used to carry out the present invention may beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withan antigen to which a monoclonal antibody to BcfA binds, collectingimmune serum from the animal, and separating the polyclonal antibodiesfrom the immune serum, in accordance with known procedures.

Monoclonal antibodies used to carry out the present invention may beproduced in a hybridoma cell line according to the technique of Kohlerand Milstein, Nature 265, 495-97 (1975). For example, a solutioncontaining the appropriate antigen may be injected into a mouse and,after a sufficient time, the mouse sacrificed and spleen cells obtained.The spleen cells are then immortalized by fusing them with myeloma cellsor with lymphoma cells, typically in the presence of polyethyleneglycol, to produce hybridoma cells. The hybridoma cells are then grownin a suitable media and the supernatant screened for monoclonalantibodies having the desired specificity. Monoclonal Fab fragments maybe produced in Escherichia coli by recombinant techniques known to thoseskilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).

Antibodies specific to BcfA can also be obtained by phage displaytechniques known in the art.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor Interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogenous Specific Binding Assay Employing a Coenzyme asLabel.” Applicants specifically intend that the disclosures of all U.S.Patent references cited herein be incorporated herein by reference intheir entirety.

Antibodies as described herein may be conjugated to a solid supportsuitable for a diagnostic assay (e.g., beads, plates, slides or wellsformed from materials such as latex or polystyrene) in accordance withknown techniques, such as precipitation. Antibodies as described hereinmay likewise be conjugated to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase,alkaline phosphatase), and fluorescent labels (e.g., fluorescein) inaccordance with known techniques. The term “antigenic equivalents” asused herein, refers to proteins or peptides which bind to an antibodywhich binds to the protein or peptide with which equivalency is soughtto be established. Antibodies which are used to select such antigenicequivalents are referred to as “selection antibodies” herein.

4. Utility

Antigens of the present invention (BcfA and fragments thereof) andformulations of such antigens are useful for producing an immuneresponse against said antigen in a mammalian subject. Such an immuneresponse is useful for the production of antibodies, which antibodiescan be used for diagnostic purposes (in detecting the presence ofBordetella) or for therapeutic purposes in treating Bordetella bypassive immunity as described herein.

Antigens of the present invention are also useful as vaccines forproviding protective immunity in mammalian subjects against Bordetellainfection.

Example 1 Passive Immunization

Groups of five C57/BL6 mice were separately injected intraperitoneallywith 200 μl of sera harvested from wild-type inoculated rats,BcfA-specific polyclonal serum, preimmune sera or sterilephosphate-buffered saline (PBS). Three to four hours after inoculation,these mice were intranasally challenged with 5×10⁵ colony forming units(cfus) of wild-type B. bronchiseptica strain RB50 in a 25 μl droplet.Seven days post-inoculation, mice were sacrificed and trachea, nasalseptum and lungs were harvested in sterile PBS and homogenized.Colonization of these organs was quantified by plating differentdilutions of the homogenate in BG blood plates containing 50 μg/ml ofstreptomycin and subsequent colony counting. The results of thisanalysis indicated that anti-serum raised against BcfA was able toprotect mice against subsequent challenge with B. bronchiseptica.

Example 2 BcfA Epitopes

The purified BcfA protein migrates in an SDS-polyacrylamide gel at amobility corresponding to ≈100 kDa, which is consistent with theannotated length (969 amino acids; FIG. 2A) of the BcfA open readingframe. BcfA displays homology to other bacterial proteins including BipAfrom Bordetella, invasins from Yersinia, and intimins fromenteropathogenic E. coli. Based on the known structure of theseproteins, the C-terminal 508 amino acid residues of BcfA are expected toencompass the extracellular region of BcfA and thus will interact withthe immune system. Accordingly, amino acid residues 461-969 of BcfA (SEQID NO:3; FIG. 2B), or one or more fragments thereof, are expected toelicit an immune response against B. bronchiseptica. Exemplary fragmentsof BcfA are shown in FIGS. 2C-2L.

Additional fragments of BcfA include antigenic regions of the BcfAextracellular domain as well as fragments expected to bind to majorhistocompatibility complex (MHC) class I and MHC class II molecules.Accordingly, the amino acid sequence of the extracellular region of BcfAwas analyzed using two independent web-based algorithms that predictantigenic sites in proteins (Table 1) and potential binding to MHC classI and MHC class II molecules (Table 2).

Multiple peptides within the extracellular region of BcfA were predictedto be antigenic and exhibit high binding affinity for human HLAmolecules (Table 3).

TABLE 1 SEQ ID Antigenic Predicted Epitope NO: Location Score'¹GDYPVTLVLED 14 443 1.200 GGPVKRPYHDIFVPVPPTVEVATD 15 165 1.179 APTVVLHT16  91 1.165 QTLLGGKIRLLRPVARLLLSP 17 350 1.162 SGVVTVTGY 18 277 1.143PQTAALLAAIKLHDPN 19 402 1.137 GKAPVVPGANGV 20 474 1.128GKPVRRPYVDTVAPTPMKVTID 21 248 1.119 GTGVVTVT 22  11 1.110 ASGPIVAIA 23 57 1.108 TMVLKVTGS 24 463 1.106 GGSLLIG 25 495 1.095 VGGSTVTVTFP 26 2871.093 RAKVKVDFP 27 200 1.092 GGDIVVTQ 28 233 1.089 GAVRTH 29   4 1.086LDGIVARF 30 387 1.086 GDVVAG 31  37 1.081 SGRVTVSGK 32 188 1.079 KEVVAGP33 127 1.078 RTVQYD 34  76 1.078 FTVASKGDV 35  47 1.073 PAGPIRVSAR 36321 1.068 DHYLDA 37 341 1.052 GAKVRID 38  24 1.051 YTVTST 39 312 1.050DITVSGT 40 149 1.037 Location is the position of the first residue.¹Score obtained using the Antigenic program which employs the method ofKolaskar and Tongaonkar (1990). FEBS Letters 276: 172-174.

TABLE 2 SEQ Predicted ID HLA BIMAS Epitope NO: Location MoleculeScore^(1,2) RRTVQYDDR 41  75 HLA-B_2705 3000 LRPVARLLL 42 360 HLA-B_27052000 AREATTMVL 43 458 HLA-B_2705 2000 IRLLRPVARL 44 357 HLA-B_2705 2000AREATTMVLK 45 458 HLA-B_2705 2000 KRPYHDIFV 46 166 HLA-B_2705 1800RRTVQYDDRV 47  75 HLA-B_2705 1800 GPVKRPYHDI 48 163 HLA-B_5102 1320EVATDSSSGR 49 181 HLA-A68.1 1200 RPYHDIFVPV 50 167 HLA-B_5102 1100IRLLRPVAR 51 357 HLA-B_2705 1000 ARFEPANGA 52 392 HLA-B_2705 1000IRVSARGPR 53 325 HLA-B_2705 1000 VRIDFPDGTF 54  27 HLA-B_2705 1000VPVPPTVEV 55 174 HLA-B_5102  660 APVVPGANGV 56 476 HLA-B_5102  660LESNKMFIYL 57 420 HLA-B60  640 GRPGDTIRV 58 111 HLA-B_2705  600MRTDGNSGV 59 271 HLA-B_2705  600 RRPYVDTVA 60 252 HLA-B_2705  600VRRPYVDTV 61 251 HLA-B_2705  600 YRATSDGDV 62 223 HLA-B_2705  600VRTHPGTGV 63   6 HLA-B_2705  600 MRTDGNSGVV 64 271 HLA-B_2705  600VRTHPGTGVV 65   6 HLA-B_2705  600 NRVPNGDYPV 66 438 HLA-B_2705  600ARLLLSPGSM 67 364 HLA-B_2705  600 YRLESNKMFI 68 418 HLA-B_2705  600FPGGTSKTV 69 207 HLA-B_5102  586 APTPMKVTI 70 260 HLA-B_5101  484GPSLGGSLLI 71 491 HLA-B_5102  484 GPSLGGSLLI 71 491 HLA-B_5101  440SPGSMTYTEI 72 369 HLA-B_5101  440 GPVKRPYHDI 48 163 HLA-B_5101  440APTPMKVTI 70 260 HLA-B_5102  440 SPGSMTYTEI 72 369 HLA-B_5102  440VVAGPDGTYR 73 129 HLA-A68.1  400 FPGGTSKTV 69 207 HLA-B_5101  381FPDGTTKEVV 74 121 HLA-B_5101  381 RESPRRTVQY 75  71 HLA-B_4403  360FPDGTTKEV 76 121 HLA-B_5101  346 AALLAAIKL 77 405 HLA-B_5102  330MPGAAGKPV 78 243 HLA-B_5101  315 FPDGTFGDV 79  31 HLA-B_5101  315VAPTPMKVTI 80 259 HLA-B_5101  315 KLHDPNYRL 81 412 HLA-A_0201  307DAWTKQTLL 82 345 HLA-B_5102  303 DTMNSDPYNR 83 430 HLA-A68.1  300YRLESNKMF 84 418 HLA-B_2705  300 GRVTVSGKGR 85 103 HLA-B_2705  300Location is the position of the first residue. ¹Score obtained using theBIMAS program developed by Parker, et al. (1994) J. Immunol. 152: 163,which provides the rank potential of 8-mer, 9-mer, or 10-mer peptidesbased on a predicted half-time of dissociation to HLA class I molecules.²Minimum scores 300 on the BIMAS site were used.

TABLE 3 SEQ Predicted ID Loca- Antigenic BIMAS Epitope NO: tion ScoreScore  RTVQYD 34  76 1.179 RRTVQYDDR 41  75 RRTVQYDDRV 47  75 1800GGPVKRPYHDIFVPVPPTVEVATD 15 165 1.179     KRPYHDIFV 46 166 1800     RPYHDIFVPV 50 167             VPVPPTVEV 55 174GKPVRRPYVDTVAPTPMKVTID 21 248 1.119    VRRPYVDTV 61 251     RRPYVDTVA 60252            VAPTPMKVTI 80 259             APTPMKVTI 70 260  315QTLLGGKIRLLRPVARLLLSP 17 350 1.162        IRLLRPVARL 44 357       IRLLRPVAR 51 357           LRPVARLLL 42 360 2000 PQTAALLAAIKLHDPN19 402 1.137    AALLAAIKL 77 405  330 GKAPVVPGANGV 20 474 1.128  APVVPGANGV 56 476  660

Example 3 Active Immunization

FIG. 3 shows that Immunization with BcfA protects mice against B.bronchiseptica challenge. Mice were immunized intraperitoneally at 0 and3 weeks with either 10 or 30 μg of BcfA adsorbed to alum or alum only.One week after the second immunization, mice were intranasallychallenged with 5×10⁵ CFU or RB50 in a 25 μl volume. Mice weresacrificed at 1 day (FIG. 3A) and 6 days (FIG. 3B) post-challenge andthe number of CFU was determined in the nasal septum, trachea and lungs.Individual symbols represent a single mouse. The dashed line representsthe lower limits of CFU detection. Black bars represent meancolonization of respective groups. A statistical analysis was carriedout using an unpaired two-tailed Student t test. The asterisks indicatethe range of the different P values (one asterisk, ≦0.05; two asterisks,≦0.005 and three asterisks, ≦0.0005).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of producing an immune response ina mammalian subject in need thereof, comprising administering to saidsubject an isolated protein or peptide comprising (i) the Bordetellacolonization factor A (BcfA) protein of SEQ ID NO: 2 or (ii) andantigenic fragments thereof comprising 20 or more contiguous amino acidsthereof, in an amount effective to produce an immune response.
 2. Themethod of claim 1, wherein said protein or peptide is an antigenicfragment of BcfA having the sequence given herein as SEQ ID NO: 3 or anantigenic fragment comprising 20 or more contiguous amino acids thereof.3. The method of claim 1, wherein said administering further comprisesan adjuvant.
 4. The method of claim 1, wherein said administeringdelivers said BcfA protein or peptide by a mucosal route.
 5. The methodof claim 1, wherein said administering delivers said BcfA protein orpeptide by a parenteral route.
 6. The method of claim 1, wherein saidadministering delivers said BcfA protein or peptide by a transdermalroute.
 7. The method of claim 1, wherein said administering deliverssaid effective amount of BcfA protein or peptide in a single doseschedule.
 8. The method of claim 1, wherein said administering deliverssaid effective amount of BcfA protein or peptide in a multiple doseschedule.
 9. A method of producing an immune response in a mammaliansubject in need thereof, comprising administering to said subject anisolated Bordetella colonization factor A (BcfA) peptide comprising anantigenic fragment of 20 or more contiguous amino acids of SEQ ID NO: 2in an amount effective to produce an immune response.
 10. The method ofclaim 9, wherein said administering further comprises an adjuvant. 11.The method of claim 9, wherein said administering delivers said BcfApeptide antigenic fragment by a mucosal route.
 12. The method of claim9, wherein said administering delivers said BcfA peptide antigenicfragment by a parenteral route.
 13. The method of claim 9, wherein saidadministering delivers said BcfA peptide antigenic fragment by atransdermal route.
 14. The method of claim 9, wherein said administeringdelivers said effective amount of BcfA peptide antigenic fragment in asingle dose schedule.
 15. The method of claim 9, wherein saidadministering delivers said effective amount of BcfA peptide antigenicfragment in a multiple dose schedule.